Susceptibility of some rodent species to monkeypox virus, and course of the infection*

Susceptibility of some rodent species to monkeypox virus, and course of the infection* S. S. MARENNIKOVA 1 & E. M. SELUHINA2 The authors studied the susceptibility of five species of rodent to monkeypox virus inoculated by various routes and the course of the infection. Reactions variedfrom complete resistance to lethal generalized infection with rash. Rabbits and white mice appeared to be the most susceptible species andyoung animals were more susceptible than adults. Monkey- pox virus was found to infect young animals by natural routes, i.e., per os and intranasally. Transmission by contact occurred among 10-day-old rabbits. Since antibodies to monkeypox virus may persistfor over a year in the sera ofconvalescent animals, serological examination of animals is recommended for studying the ecology of this virus. When it was discovered that monkeypox virus can induce a pox-like infection in man (2, 7) numerous problems remained to be solved, since the ecology of the agent had not been adequately studied. It has still not been determined for certain what is the source of human infection and whether monkeys are the only carrier of this virus. During the outbreak of monkeypox in Rotterdam Zoo (3) it was observed that not only primates but also the giant ant-eater can be infected with this virus. It therefore seemed expedient to study the susceptibility of various animal species to monkeypox virus and to determine the pecularities of the course of infection. MATERIALS AND METHODS Viruses The reference strain of monkeypox virus Copen- hagen was used in the form of a chorioallantoic membrane (CAM) culture from the 7th-1 5th passages on chick embryos. The infective dose varied from 101 to 102 pock-forming units (PFU), depending on the species of animal and the aim of the experi- ment. * From the Laboratory of Smallpox Prophylaxis, Research Institute of Virus Preparations, Ministry of Health (WHO Collaborating Centre for Smallpox and other Poxvirus Infections), Moscow, USSR. The data given in this paper were presented at the WHO Informal Consultation on Monkeypox and Related Poxviruses, Geneva, December 1973. Chief. Senior research worker. Animals Adult rabbits (chinchilla strain) weighing 2.5- 3.0 kg and 10-day-old rabbits; guinea-pigs weighing 250-300 g; 3-week-old hamsters; adult and newborn white rats; and white mice aged 1-2, 8, 12, and 15 days were used in the experiments. Methods of inoculation Routine methods were used to inoculate animals by the intracerebral, intravenous, intracardial, intra- peritoneal, intranasal, and intradermal routes, and on scarified skin. For foot-pad inoculation of guinea- pigs and white mice, 0.1 ml and 0.01 ml of virus suspension, respectively, were used. A special needle was used to infect mice per os. Young rabbits were infected with a probe or by adding the virus to the milk on which they fed. Adult rabbits were infected by means of a pipette. Virus isolation from the blood and viscera At the peak of the disease or 1, 2, 3, 4, and 5 weeks after inoculation, some of the animals from each group were killed and their blood and viscera (lungs, liver, kidneys, spleen, lymph nodes, brain, and testicles) were collected under aseptic conditions. To isolate the virus from these organs, the latter were ground in a mortar and 20% suspensions (by weight) in McIlvain buffer were prepared. Centrifugation was not used, but before inoculation the suspensions were shaken vigorously. The suspensions were tested by inoculation on CAM of 12-day-old chick embryos. These were opened after 72 h of incubation at 35°C. 3413 - 13 - BULL. WORLD HEALTH ORGAN., Vol. 53, 1976 S. S. MARENNIKOVA & E. M. SELUHINA If there were no lesions on CAM or if their morpho- logy was doubtful, another passage was performed with a CAM suspension as the inoculum. If the inoculum produced lesions typical of monkeypox virus, the results were considered positive. The blood for virus isolation was diluted 1: 3 in distilled water. The viscera and blood were tested either immediately after collection or after storage at 4°C for 1-3 days. Sera for testing were prepared by the standard method. Serological tests The haemagglutination inhibition test was per- formed with 2 and/or 4 haemagglutination (HA) units of vaccinia virus and a 1% suspension of rooster red cells. Virus neutralization and precipita- tion tests were carried out as previously described (1, 8). Sera were diluted 1: 5 and then heated at 65°C for 20 min before testing. RESULTS The results of the experiments performed on each species of animal are given below. Rabbits The susceptibility of rabbits to monkeypox virus was seen to depend on the route of inoculation and the age of the animal. Intravenous inoculation of adult animals with the virus in a dose of 107 PFU caused a severe generalized process with fever from the 3rd to the 7th day, conjunctivitis, rhinitis, extensive rash on the skin and mucous membranes, and loss of weight. The rash was observed 5-6 days after inoculation and had the appearance of papules, which developed later into pustules (Fig. 1). In some cases the lesions became haemorrhagic. As a rule, the formation of crusts began on about the 8th day. After a fortnight the crusts fell off and the scabbing stage ended within 3 weeks. Eleven out of twelve rabbits survived the infection and one died of cachexia a month later. During the first days of the disease the virus could be isolated from the blood. Later, it was isolated from some lymph nodes and from the kidneys (7th day of the disease). The virus was also detected in the tissue of the testicles in one of three convalescent animals 22 days after inocula- tion (neither crusts nor other signs of the disease were observed). The rabbits inoculated by the intra- venous route developed antibodies 7 days after infection. By the 14th day the antihaemagglutinin titre rose to 128-256 and persisted at the level of 40-80 for more than 12 months. Virus neutralizing antibodies in a titre of 640 and precipitating anti- bodies were also detected in the sera taken more than a year later. Rabbits infected on scarified skin with a virus dose of 105 and 106 PFU per 0.1 ml developed a localized papulopustular eruption at the inoculation site. In some cases the infection was followed by generaliza- tion of the process (fever, rash on the skin and mucous membranes). Intradermal inoculation of an equal dose of the virus induced dense infiltrates with necrosis and haemorrhages in the centre. No clinical reaction was observed in adult rabbits infected per os, even with a high concentration of the virus (1.4 x 109 PFU per 2 ml). An increase of virus neutralizing antibodies (up to 20) was observed 25 days after oral inoculation in one of two animals. Young rabbits appeared to be much more sus- ceptible to monkeypox virus: the 10-day-old rabbits infected per os with a dose of 106-107 PFU per ml of virus developed an acute generalized process with rash. Adynamia, loss of appetite, and diarrhoea appeared 4-6 days after oral inoculation. Eruptions on the inner side of the ears and around the lips and nose were observed in most of the animals, with subsequent suppurative conjunctivitis and rhinitis and rash spreading over the body (Fig. 2). The disease was accompanied by considerable loss of weight and as a rule ended in death 4-14 days after inoculation. At the acute stage of the disease the virus could be isolated easily from the viscera, and it was isolated from the blood (a few pocks on CAM), lungs, liver, and spleen (confluent pocks on CAM inoculated with a 20%. suspension of those organs) of a young rabbit killed at the acute stage of the disease (4th day after inoculation). On the 7th day, the virus could be isolated from the lungs (in a titre of > 105 PFU per ml) and kidneys (in a titre of 2.6 x 103 PFU per ml), but an attempt to isolate it from the blood was unsuccessful. The 10-day-old rabbits were also found to be highly susceptible to intranasal inoculation. In the course of the disease they lost appetite and weight, the disease terminating in death within 4-5 days. No rash appeared. When uninfected animals and animals infected either per os or intranasally were kept together (the whole litter was kept in one cage together with the mother), the infection was trans- mitted by contact: 4 uninfected young rabbits sickened and 3 of them died 14 days after the experiment started and 6-8 days after the first signs of infection became evident in inoculated animals. 14 MONKEYPOX VIRUS INFECTION IN RODENTS Table 1. Susceptibility of some rodent species to monkeypox virus Seis Inoculation Age or Mortality AntibodySpecies route weight Symptoms formAtionNo. a % fomtn Rabbit intravenous general disease with fever and 1/12 8 + rash per os adult, 2.5- none 0/2 0 ± b intradermal 3.0 kg dense infiltration with necrosis 0/10 0 + on scarified skin local eruption followed by 0/10 0 + generalization intranasal loss of weight, adynamia 5/6 83 + 10 d per os acute general disease with rash 17/20 85 + White intranasal 8-15 d loss of weight, adynamia 17/17 100 n.d. mouse 8 d loss of weight, adynamia 4/10 40 n.d. per os 12 d loss of weight, adynamia 7/29 24 + intraperitoneal 8 d loss of weight, adynamia 10/10 100 n.d. intradermal 8 d local infiltration 5/10 60 n.d. 8 d foot oedema, general disease 18/18 100 n.d. foot pad 12 d foot oedema, general disease 3/5 60 n.d. White rat intranasal none 0/6 0 n.d. intravenous adult none 0/6 0 n.d. on scarified none 0/6 0 n.d. skin intranasal 1-3 d adynamia 24/24 100 n.d. Guinea-pig intracardial none 0/5 0 + intranasal none 0/5 0 + 250-300 g per os none 0/5 0 foot pad foot oedema 0/5 0 + Hamster on scarified none 0/6 + skin intranasal 3 weeks none 0/6 + per os none 0/3 + intracardial none 0/75 + a Numerator = number of dead animals; denominator = total number of animals inoculated. b Antibody formation in one out of two animals. 15 S. S. MARENNIKOVA & E. M. §ELUHINA A high concentration of virus (102 PFU per ml) was detected in the viscera of the dead animals. The mother rabbit had no symptoms of the disease, but haemagglutination-inhibiting antibodies (in a titre of 80 with 4 HA units) and neutralizing and precipitating antibodies were found in her serum 28 days after the young rabbits were inoculated with virus. Different types of antibodies to vaccinia virus were also found in the serum of a surviving young rabbit (virus neutralizing, precipitating, and haemag- glutination-inhibiting antibodies in a titre of 160 with 4 HA units). Guinea-pigs The susceptibility of guinea-pigs was determined by per os, intranasal, intracardial, and foot-pad inoculation of the virus. No clinical reaction was observed in animals infected by these methods, except for foot oedema after foot-pad inoculation. Seven days after inoculation virus could be detected only in the lungs of those guinea-pigs that had been inoculated by the intracardial route. After a fortnight the virus was not traceable in any of the animals infected by the above-mentioned methods. By that time haemagglutination-inhibiting antibodies in a titre of 16-64 were observed in the sera of guinea-pigs inoculated by the intranasal and intracardial routes and into the foot-pad. White rats No pathological symptoms developed in adult white rats infected by the intravenous or intranasal routes, or on scarified skin. The virus was not isolated from the blood and viscera of rats infected by the intranasal and intravenous routes, and it did not multiply in the skin of rats infected on scarified skin. Different results were obtained by infecting newborn rats (1-3 days old). These rats were sensitive to monkeypox virus inoculated by the intranasal route, since they developed disease terminating in death on the 5th or 6th day. The virus was isolated from their lungs and liver in a titre of 102-103 PFU per ml. White mice White mice were shown to be highly susceptible to monkeypox virus inoculated by various routes. When 8-day-old white mice were inoculated by the intraperitoneal or intranasal route in doses of 1.2 x 106 PFU and even into the foot-pad (with a dose of 6 x 102 PFU) they developed disease the main symptoms of which were flabbiness, loss of appetite, and oedema of the foot (after foot-pad inoculation). All these mice died. Intradermal inoculation of the virus into 10 mice resulted in infiltrates with the death of 5 mice (50%/). Mice inoculated by the oral route became flabby and lost appetite, and 40%. of them died. Comparison of the susceptibility of 8-day-old mice to oral and to intranasal inoculation showed that the LD50 was lower when the virus was inoculated by the intranasal route. Higher sensitivity of white mice to intranasal infection was demonstrated by the inoculation of older animals. The 12-day-old mice infected per os sickened and died in only 14% of cases. On the other hand, 100% mortality was observed in 15-day-old mice after intranasal inoculation with the same dose of virus. The virus could be isolated (after oral inoculation) from the blood after one week and from the viscera (lungs, liver, spleen, and kidneys) after 3 weeks. The testing of sera from convalescent mice 42 days after infection per os revealed the presence of haemagglutination-inhibiting antibodies in a titre of > 256. A considerable amount of virus was detected in the lungs and other organs at the acute stage of the disease as a result of intranasal inoculation. Hamsters The animals were infected per os, by the intranasal and intracardial routes, and on scarified skin with a dose of 1.5-5.9 x 107 PFU. No symptoms of the disease were observed in these hamsters. Never- theless, in animals infected by the intracardial route, the virus was detectable in the lungs, liver, and spleen during the first week after inoculation and in the kidneys during the first 3 weeks. Furthermore, despite the absence of clinical signs of the disease, marked pathological alterations were observed in the viscera of hamsters inoculated by the intracardial route. Those findings will be the subject of a separate report. DISCUSSION The pathogenicity of monkeypox virus for white mice inoculated by the intranasal and intracerebral routes and for rabbits inoculated by the intradermal, cutaneous, subcutaneous, intracerebral, intravenous, and intratesticular routes, as well as the resistance of guinea-pigs to the virus inoculated by the intra- venous, intraperitoneal, and subcutaneous routes, were reported by von Magnus et al. (5), Prier & Sauer (9), Gispen et al. (4), and Marennikova 16 :.: x a) * i E .C 0.s ... * 0::: .l.Ca). *:C.o *C. .a) . CoC . -o Oll Fig. 2. Generalized monkeypox infection in a 10-day-old rabbit: (a) general view; (b) inner surface of ear. MONKEYPOX VIRUS INFECTION IN RODENTS et al. (6). The susceptibility of hamsters had not been studied before. The observations mentioned above were con- firmed in our present study. These authors merely reported the pathogenicity of the virus, without details of the course of infection. Our task was to investigate the character of the infection process, the distribution of virus in the blood-stream and internal organs, and the immunological response after virus inoculation by various routes. Special attention was paid to the intranasal, per os, and contact routes, which are the possible ways of virus spread in nature, since they had never been studied previously, except for the intranasal route in mice. The studies, performed with 5 species of rodent, showed that monkeypox virus can induce different kinds of reaction, ranging from complete resistance to a generalized lethal infection, depending on the species ofanimal, its age, and the route ofinoculation. In our experiments, the disease developed in sus- ceptible species (white mice and young rabbits) with infection methods that could be the ways in which the disease is transmitted in natural conditions (per os, intranasal infection). Generalized infection of rabbits with extensive eruptions on mucous mem- brane and skin permitted the release of the virus into the environment and the transmission of infection by contact. The susceptibility of animals to monkeypox virus varied substantially depending on age. This was best demonstrated in white rats. Adult rats appeared to be resistant to large doses of virus inoculated by different routes, while newborn animals (1-3 days old) became sick on the 5th or 6th day after intranasal inoculation. The same effect was observed in rabbits. While adult rabbits developed no overt infection after per os inoculation, young rabbits inoculated by the same route responded with generalized infection with skin eruption and a high mortality rate. The possibility that infection was transmitted by contact (from infected to uninfected young rabbits) was established. This fact is particularly interesting as it indicates the possibility of horizontal transmission of monkeypox among animal species (e.g., rodents). All the isolates of variola-like virus and one of monkeypox virus (strain 9411) were obtained from the kidneys of wild monkeys or apparently healthy laboratory monkeys. This fact served as a basis for studies of virus behaviour in animals with various types of infectious process. geluhina et al. (10) observed that virus was continuously excreted by persons convalescing from smallpox. Despite the generalized type of infection, the virus could not be detected in rabbit kidney after the 7th day, although it was detected in testicle tissue for 22 days. On the other hand, the virus was found in hamster kidney for 1-3 weeks (though attempts to isolate it from other organs were negative and clinical features were absent). The long persistence of pox antibodies in the sera of animals convalescent after monkeypox infection, which we observed in our studies, coincides with the observa- tion of R. Gispen et al. (unpublished observations, 1973), who found virus neutralizing and fluorescent antibodies in the sera of two convalescent orang- utans for more than 5 years. On the basis of our findings, serological examination of susceptible animals can be recommended for ecological studies of monkeypox virus. The results presented here show that it was expedient to study the ecology of monkeypox virus and that it is necessary to investigate animal species other than monkeys in areas where monkeypox infection of man has been reported. RIESUMI! SENSIBILITE DE QUELQUESE5PtCES DE RONGEURS AU VIRUS DU MONKEYPOX ET E'VOLUTION DE L'INFECTION On a eprouve la sensibilite au virus du monkeypox de cinq especes de rongeurs de laboratoire en le leur ino- culant par differentes voies. Les reactions allaient de la resistance presque complete a l'infection lethale gen&e ralisee selon l'espece de l'animal, son age et la voie d'inoculation. Les jeunes lapins et les souris blanches de 8 a 12 jours etaient les plus sensibles: ils pr6sentaient une infection g6n6ralisee apres inoculation intranasale et per os. L'infection pouvait etre transmise par contact parmi les rats de 10 jours. I1 a ete demontre que le virus du monkeypox etait capable d'infecter des animaux par les voies naturelles de transmission virale. En raison de la persistance des anticorps specifiques du monkeypox dans le serum des animaux convalescents, il est recom- mande de recourir aux examens s6rologiques pour etudier la distribution du virus du monkeypox dans la nature. 2 19 20 S. S. MARENNIKOVA & E. M. SELUHINA REFERENCES 1. BOULTER, E. A. Journal of hygiene, 55: 502 (1957). 2. FOSTER, S. 0. Bulletin of the World Health Organ- ization, 46, 569 (1972). 3. GISPEN, R. & KAPSENBERG, J. G. 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